Conventional absorption spectroscopy is widely applied to probe constituents in the liquid phase. In conjunction with several separation techniques, including High-Performance Liquid Chromatography (HPLC) and Capillary Electrophoresis (CE), absorption spectroscopy has become an indispensable tool in virtually all fields of chemistry. It has been used to provide quantitative determination of many important chemical compounds including species as varied as nucleic acids, amino acids, proteins, carbohydrates, terpenoids, steroids, antibiotics, pharmaceuticals, pesticides, hydrocarbons, and a host of inorganic substances. Most of these analyses have involved using a HPLC or CE column to separate the species and then an absorption detector to measure the species' concentration as they elute off the column. The vast majority of research has involved developing specialized columns for various applications.
There has been some progress in increasing the sensitivity of these absorption detectors, which has mostly focused on using other analytical techniques to measure concentration. These techniques, including fluorescence, electrochemical detection, and mass spectrometry, have increased the sensitivity of HPLC and CE by about a factor of 100-1000. However, these advances have also limited the number of applications and increased complexity. For example, fluorescence detection requires that the molecules of interest be “tagged” with an appropriate dye, electrochemical detection requires that the analyte undergo a redox reaction, and mass spectrometry requires extensive equipment and specialized couplings between the column and the detector. Due to these complexities, absorption is still the most commonly used HPLC and CE detection method. Likewise standard techniques like colorimetry rely on using absorption spectroscopy to detect pH, metal contamination in wastewater, and many titration products.
In conventional absorption spectroscopy, a light source is passed through an absorbing sample. The intensity of the light is measured before, I0, and after, I, the absorbing media. The concentration of the absorbing species can be determined from Beer's Law:I/I0=e−Lσc∝1−Lσc where L is the sample length, σ is the molecule's absorption cross section, and c is the species' concentration. The minimum detectable concentration of the absorbing species depends on how accurately one can measure the change in light intensity (ΔI=I−I0). For typical absorption experiments, ΔI/I0˜10−4 after one second of averaging. Absorption spectroscopy is an attractive analytical tool because it is inexpensive, simple, and provides an absolute concentration without calibration. Unfortunately, conventional absorption spectroscopy does not provide the sensitivity necessary for many analytical applications.
The sensitivity of absorption spectroscopy can be increased by either measuring ΔI more accurately or by use of a longer sample path length. The latter can be simulated by passing the light through the liquid cell several times (multi-pass cell) to increase the path length by an order of magnitude. One of the most common methods employed to measure AI more accurately in liquids is photoacoustic spectroscopy (U.S. Pat. No. 4,303,343), which relies on measuring the acoustic wave generated by absorption in liquids. Although this technique can be very sensitive due to its background-free detection method, it requires very high-power lasers (1 Watt) to achieve the sorts of sensitivities described in this patent and is therefore not widely applicable. Another proposed technique utilizes thermal-lensing effects in liquid samples to measure weak absorptions (U.S. Pat. No. 4,544,274) by placing the liquid cell within a laser cavity. This method is also limited in utility due to its complexity and specialized equipment.
World Precision Instruments supplies a commercial absorption spectroscopy instrument based on a liquid waveguide capillary cell (cf., U.S. Pat. Nos. 5,444,807; 5,570,447; and 5,604,587). The instrument uses a 1 to 10 meter long, hollow capillary tube made of (or coated with) Teflon AF. The tube is filled with liquid and light is coupled into the capillary. Similar to an optical fiber, the light is confined within and propagates down the length of the tube. The net absorption is measured by comparing the light intensity before and after the capillary. An advantage of this technology is that a small sample volume (200 microliters per meter of tubing) can extend over a very long light path. The system can use very robust, inexpensive parts, e.g., LEDs or lamps for the light source. However, for some analytical separation techniques, including HPLC and CE, the resolution (ability to distinguish separated compounds) is directly dependent on the volume of the analyzer and the capillary length, and thus much smaller liquid volumes than those used in this instrument are needed.
In 1988, Anthony O'Keefe and David A. G. Deacon introduced the idea of Cavity Ring Down Spectroscopy (CRDS). (“Cavity Ring-Down Optical Spectrometer for Absorption Measurements Using Pulsed Laser Sources”, Review of Scientific Instruments, vol. 59, no. 12, December 1988, pp. 2544-2551) In CRDS, light passes through the mirror in order to enter the cavity and is detected by passing through the output mirror. Unlike multi-pass methods, there is not an entrance or exit hole in either mirror. If the light is of the correct frequency, it constructively interferes with itself and builds up in the optical cavity. When the intensity has built up sufficiently, the laser is rapidly blocked and the decay of light intensity out of the cavity is measured. If the system is well aligned, this decay will be exponential with a time constant, τ, which depends on all of the losses in the cavity. These losses include mirror transmission (typically on the order of 100 ppm) and any absorption or scatter in the cavity. The major advantage of CRDS is that the effective path length of the absorbing species is dependent on the reflectivity of the mirrors, Leff=L/(1−R). In a one-meter long gas cell with highly reflective mirrors (R˜99.99%) the effective path length approaches 10,000 meters. This enormous enhancement in effective path length allows for very small concentrations to give rise to detectable changes in τ. Moreover, since τ is an absolute measure of cavity loss, CRDS gives an absolute concentration without need for calibration. Due to these advantages, CRDS has since led to over 300 publications worldwide, and is emerging as a powerful new analytical tool.
Unfortunately, CRDS cannot be applied effectively to liquids. The large scattering losses in liquids coupled with short cavity lengths result in a very short ringdown time that is difficult to accurately measure. Most liquid cells, especially those used in HPLC and CE, have path lengths on the order of 1-10 mm. Even with high reflectivity mirrors, the ringdown time of these cells is less than 100 nanoseconds, which is too fast to measure very accurately.
Despite these limitations, a research group at Stanford University has recently demonstrated cavity ringdown spectroscopy in a liquid cell. (A. J. Hallock, et al., “Direct Monitoring of Absorption in Solution by Cavity Ring-Down Spectroscopy”, Analytical Chemistry, vol. 74, pp. 1741-1743 (2002)) In their system, a long (−21 cm) liquid cell is bounded by two high-reflectivity mirrors. Pulsed laser light is injected through one of the mirrors and a photomultiplier tube is used to measure the transmitted intensity through the other mirror. The residence time of the light in the cavity is determined by the total loss in the system that is given by the sum of the mirror transmission and losses due to scatter and absorption in the media. Typically, these ringdown times are on the order of 400 ns and require a pulsed dye laser coupled with a fast detector and data acquisition system. The main limitation in this design is the very large liquid cell required to gain measurable cavity ringdown times (minimum volume of 5 mL). As noted above, many of the key applications of enhanced liquid absorption spectroscopy require very small sample volumes (microliters) and are therefore not amenable to this system.
A similar CRDS system for liquid samples is described in a paper by S. Xu, et al., “Cavity ring-down spectroscopy in the liquid phase”, Review of Scientific Instruments, vol. 73, pp. 255-258 (2002). In that system, a short (1 cm) liquid cell is inserted into a much longer (48 cm) optical cavity. Like all of these cavity-based systems, they pass the light multiple times through the cell containing the liquid sample, thus multiplying the 1 cm path length through the cell to an effective 900 cm length. By super-polishing the walls of the liquid cell and placing them at precisely Brewster's angle (the cell is very alignment sensitive), they can minimize scattering losses from the cell walls. In addition to requiring a specialized highly polished glass cell that is precisely placed, the cavity itself is also alignment sensitive. Any misalignment of the cavity mirrors not only decreases the accuracy of the analysis, but also changes the way the beam passes through the liquid cell, introducing additional loss. Also, if a different liquid solvent is used, with a different refractive index, the liquid cell must also be changed (cut at a different angle) in order to prevent introducing scattering loss.
Integrated Cavity Output Spectroscopy (ICOS) was developed at Los Gatos Research, Inc., Mountain View Calif., to enhance the sensitivity of absorption spectroscopy for use with very small sample volumes. (A. O'Keefe, J. J. Scherer, and J. B. Paul, “CW Integrated Cavity Output Spectroscopy”, Chemical Physics Letters, vol. 307, no. 5-6, Jul. 9, 1999, pp. 343-349). This technique is similar to CRDS in that it uses a high-finesse optical cavity, but does not involve blocking the laser or measuring cavity decay. Instead, the intensity of the beam passing through the cavity is continuously measured similar to a standard absorption experiment. The measured output varies randomly between high intensity and no intensity as the laser wanders slowly in and out of resonance with the cavity (due to mechanical and thermal drifts). In order to randomize this transmission on a faster timescale (and thus allow for faster signal averaging), the laser is dithered while the cavity length is slightly oscillated using a piezoelectric transducer. This forced fast randomization allows the cavity output to be averaged to within ΔI/I0˜10−2 in one second. Although this is significantly noisier than standard absorption, ICOS retains the long effective path length realized in CRDS. In order to calibrate ICOS, it is necessary to know the reflectivities of the mirrors, which can be determined using CRDS prior to installment or by measuring an absorption standard. Until now, ICOS has only been used with gas samples.
In 2000, Joshua Paul and Anthony O'Keefe developed Off-Axis ICOS (see assignee's pending U.S. patent application Ser. No. 09/976,549, filed Oct. 12, 2001, and incorporated by reference herein), a more sensitive variant of ICOS that does not rely on high-voltage components. The sensitivity of ICOS is limited by the large variations in output intensity due to the light constructively and destructively interfering with itself in the cavity. In order to decrease ΔI/I0, it is necessary to quell these interferences. In ICOS, these interferences were partially suppressed by dithering both the cavity and the laser. In Off-Axis ICOS, these interferences are almost completely eliminated by aligning the cavity in an off-axis configuration. This configuration, coupled with the slight astigmatism of the cavity mirrors, increases the beam's reentrant condition from a single pass (for standard ICOS) to almost 1000 passes. For most laser light sources, after the 1000 passes, the light is unable to interfere with itself because the distance it has traveled exceeds the coherence length of the laser. Thus, the cavity output is much more stable than standard ICOS and ΔI/I0˜10−4 can easily be achieved with only one second of averaging without the use of any high-voltage transducers. Moreover, since the off-axis beam path is not unique, the system is extremely insensitive to changes in alignment, making it much more robust than multi-pass cells, CRDS, or ICOS. The effective path length of Off-Axis ICOS is again related to the reflectivity of the mirrors and is typically ˜10000 times the cavity length.
An object of the present invention is to provide a cavity-based absorption spectroscopy apparatus adapted to measure liquid samples of small volume (˜microliters) with enhanced absorption sensitivity.